Methods for determining test result validity using fluid flow transit time across a test strip

ABSTRACT

The present disclosure relates to methods for determining a wavefront position of a liquid on a surface of an assay test strip placing a liquid on the surface of the test strip; and acquiring one or more signals from the surface of the test strip at one or more times, comparing the one or more acquired signals to a threshold, wherein the wavefront position is a position on the surface of the test strip where a signal is greater than or less than a threshold (e.g., fixed or dynamic threshold). Such methods may be used to determine the wavefront velocity of a liquid on a surface of an assay test strip and the transit time of a liquid sample to traverse the one or more positions on the surface of the assay test strip.

BACKGROUND

Assay test kits currently are available for testing a wide variety ofmedical and environmental conditions or compounds, such as a hormone, ametabolite, a toxin, or a pathogen-derived antigen. Most commonly thesetests are used for medical diagnostics either for home testing, point ofcare testing, or laboratory use. For example, lateral flow tests are aform of immunoassay in which the test sample flows along a solidsubstrate via capillary action. Some tests are designed to make aquantitative determination, but in many circumstances all that isrequired is a positive/negative qualitative indication. Examples of suchqualitative assays include blood typing, most types of urinalysis,pregnancy tests, and AIDS tests. For these tests, a visually observableindicator such as the presence of agglutination or a color change ispreferred.

A common problem with lateral flow test strips is that different teststrips tend to produce slightly different results. Unfortunately, no twotest strips will perform exactly alike (i.e. generate identical testresult values) even if the test strips have the same amount of reagentembedded therein, and even if they are both exposed to the same amountof analyte. Such discrepancies in lateral flow assay test results may bedue to differences in the physical properties of individual test strips,and also by differences in the fluid flow path along different teststrips.

SUMMARY

The present disclosure relates generally to methods for determining awavefront position of a liquid (e.g., a liquid test sample) on a surfaceof a test strip (e.g., lateral flow test strip). Such methods may beused to determine the transit time of a liquid across the surface of anassay test strip or the start time of an assay.

The present disclosure relates generally to methods for determining awavefront position of a liquid on a surface of a test strip by placing aliquid on the surface of the test strip; acquiring one or more signalsfrom the surface of the test strip at one or more times; and comparingthe one or more acquired signals to a threshold, wherein the wavefrontposition is a position on the surface of the test strip where a signalis greater than or less than a threshold (e.g., fixed or dynamicthreshold).

The present disclosure relates generally to methods for determining awavefront position of a liquid on a surface of a test strip by placing aliquid on the surface of the test strip; acquiring one or more signalsfrom the surface of the test strip at one or more times; comparing theone or more acquired signals to a threshold, and identifying a signalfurthest from where the liquid was placed on the surface of the teststrip where a signal is greater than or less than a threshold (e.g.,fixed or dynamic threshold).

In an embodiment, the wavefront position is any position on the surfaceof the test strip where a signal is greater than or less than athreshold. In an embodiment, the wavefront position is a position on thesurface of the test strip furthest from where the liquid was placed onthe test strip where a signal is greater than or less than a threshold.In an embodiment, the wavefront position is a position on the surface ofthe test strip where a signal is greater than or less than a thresholdand is greater or less than all other signals acquired from the surfaceof the test strip.

In an embodiment, the one or more acquired signals are subtracted from aconstant prior to being compared to the threshold. In an embodiment, theone or more acquired signals are divided by a constant prior to beingcompared to the threshold. In an embodiment, two acquired signals arecompared with each other prior to one signal being compared to thethreshold. In an embodiment, two acquired signals are subtracted fromeach other prior to one signal being compared to the threshold. In anembodiment, a first acquired signal is divided by a second acquiredsignal prior to the first signal being compared to the threshold.

In an embodiment, the signal is an image. In an embodiment, the image isa picture. In an embodiment, the image is acquired by an image-baseddetector.

In an embodiment, the test strip is a lateral flow assay test strip.

In an embodiment, the liquid is a test sample.

In an embodiment, the liquid is placed on the surface of the test stripprior to acquisition of a first signal. In an embodiment, the liquid isplaced on the surface of the test strip subsequent to acquisition of afirst signal.

In an embodiment, a signal acquired prior to the liquid being placed onthe surface of the test strip is compared to a signal acquired after theliquid has been placed on the surface of the test strip. In anembodiment, a signal acquired after the liquid being placed on thesurface of the test strip is compared to a signal acquired after theliquid has been placed on the surface of the test strip.

In an embodiment, two sequential signals acquired from the surface ofthe test strip are compared. In an embodiment, two nonsequential signalsacquired from the surface of the test strip are compared.

The present disclosure provides methods for determining a wavefrontvelocity of a liquid on a surface of a test strip by placing a liquid onthe surface of the test strip; acquiring signals from the surface of thetest strip at two or more times; comparing the one or more acquiredsignals to a threshold, identifying two signals that are greater than orless than a threshold (e.g., fixed or dynamic threshold); determiningthe distance between the two acquired signals; and calculating thewavefront velocity by dividing the distance between the two acquiredsignals by the time between acquisition of the two signals.

The present disclosure provides methods for determining a wavefrontvelocity of a liquid on a surface of a test strip by placing a liquid onthe surface of the test strip; acquiring a first signal from the surfaceof the test strip at a first time; acquiring a second signal from thesurface of the test strip at a second time; comparing the first signaland the second signal; and determining a position furthest from wherethe liquid was placed on the surface of the test strip where adifference exists between the first and second signal to be thewavefront of the liquid; calculating the amount of time that elapsedbetween acquisition of the first signal and the second signal;determining the distance traveled by the wavefront from acquisition ofthe first signal and the second signal; and calculating the wavefrontvelocity by dividing the distance traveled by the wavefront by the timebetween acquisition of the first and the second signal.

In an embodiment, the acquired signals are subtracted from a constantprior to being compared to the threshold. In an embodiment, the acquiredsignals are divided by a constant prior to being compared to thethreshold. In an embodiment, two acquired signals are compared with eachother prior to one signal being compared to the threshold. In anembodiment, two acquired signals are subtracted from each other prior toone signal being compared to the threshold. In an embodiment, a firstacquired signal is divided by a second acquired signal prior to thefirst signal being compared to the threshold.

In an embodiment, the signal is an image. In a further embodiment, theimage is a picture. In an embodiment, the image is acquired by animage-based detector.

In an embodiment, the test strip is a lateral flow assay test strip.

In an embodiment, the liquid is a test sample.

In an embodiment, the liquid is placed on the surface of the test stripprior to acquisition of the first signal. In an other embodiment, theliquid is placed on the surface of the test strip subsequent toacquisition of the first signal.

In an embodiment, a signal acquired prior to the liquid being placed onthe surface of the test strip is compared to a signal acquired after theliquid has been placed on the surface of the test strip. In anembodiment, a signal acquired after the liquid being placed on thesurface of the test strip is compared to a signal acquired after theliquid has been placed on the surface of the test strip.

In an embodiment, two sequential signals acquired from the surface ofthe test strip are compared. In an embodiment, two nonsequential signalsacquired from the surface of the test strip are compared.

The present disclosure also provides methods for determining a transittime of a liquid to cross the surface of a test strip by acquiring twoor more signals from the surface of the test strip; and determining afirst signal that is greater than or less than a threshold and a lastsignal that greater than or less than a threshold, wherein the transittime of the liquid to cross the surface of the test strip is the timebetween the acquisition of the first signal and the last signal.

In an embodiment, the first signal is acquired from the surface of thetest strip. In an embodiment, the first signal is the insertion of theassay test strip into a reader or any mechanical interaction of the teststrip with a reader.

The present disclosure also provides methods for determining a transittime of a liquid to advance from a first position on a surface of a teststrip to a second position on a surface of a test strip by calculatingthe wavefront velocity of the liquid as described by the methods of thepresent disclosure; and determining the transit time of the liquid tocross the surface of the test strip by multiplying the wavefrontvelocity by the distance separating the first position and secondposition on the surface of the test strip.

The present disclosure also provides methods for determining a transittime of a liquid to advance from a first position on a test strip to asecond position on a surface of an test strip by determining thewavefront velocity of the liquid, comprising acquiring two or moresignals from the surface of the test strip at two or more times;identifying at least two signals that are greater than or less than athreshold (e.g., fixed or dynamic threshold); determining the distancebetween the two or more signals; and calculating the wavefront velocityby dividing the distance between the two or more signals by the timebetween acquisition of the two or more signals; and calculating thetransit time of the liquid to advance from the first position to thesecond position on the test strip by multiplying the wavefront velocityby the distance separating the first position and second position on thetest strip.

The present disclosure also methods for determining a transit time of aliquid to advance from a first position on a test strip to a secondposition on a surface of an test strip by determining the wavefrontvelocity of the liquid, comprising placing a liquid on the surface ofthe test strip; acquiring a first signal from the surface of the teststrip at a first time; acquiring a second signal from the surface of thetest strip at a second time; comparing the first signal and the secondsignal; and determining a position furthest from where the liquid wasplaced on the surface of the test strip where a difference existsbetween the first and second signal to be the wavefront of the liquid;calculating the amount of time that elapsed between acquisition of thefirst signal and the second signal; determining the distance traveled bythe wavefront from acquisition of the first signal and the secondsignal; and calculating the wavefront velocity by dividing the distancetraveled by the wavefront by the time between acquisition of the firstand the second signal; and calculating the transit time of the liquid toadvance from the first position to the second position on the test stripby multiplying the wavefront velocity by the distance separating thefirst position and second position on the test strip.

The present disclosure also provides methods for determining a startingtime for a test assay by placing a liquid on the surface of the teststrip; acquiring one or more signals from the surface of the test stripat one or more times; comparing the one or more acquired signals to athreshold, wherein the starting time is determined as the time at whicha first signal is acquired that is greater than or less than athreshold.

In an embodiment, the one or more acquired signals are subtracted from aconstant prior to being compared to the threshold. In an embodiment, theone or more acquired signals are divided by a constant prior to beingcompared to the threshold. In an embodiment, two acquired signals arecompared with each other prior to one signal being compared to thethreshold. In an embodiment, two acquired signals are subtracted fromeach other prior to one signal being compared to the threshold. In anembodiment, a first acquired signal is divided by a second acquiredsignal prior to the first signal being compared to the threshold.

In an embodiment, the signal is an image. In a further embodiment, theimage is a picture. In an embodiment, the image is acquired by animage-based detector.

In an embodiment, the test strip is a lateral flow assay test strip.

In an embodiment, the liquid is a test sample.

In an embodiment, the methods further comprise acquiring a third imagefrom the surface of the test strip at a third time.

In an embodiment, the liquid is placed on the surface of the test stripprior to acquisition of the first signal. In another embodiment, theliquid is placed on the surface of the test strip subsequent toacquisition of the first signal.

In an embodiment, a signal acquired prior to the liquid being placed onthe surface of the test strip is compared to a signal acquired after theliquid has been placed on the surface of the test strip. In anembodiment, a signal acquired after the liquid being placed on thesurface of the test strip is compared to a signal acquired after theliquid has been placed on the surface of the test strip.

In an embodiment, two sequential signals acquired from the surface ofthe test strip are compared. In an embodiment, two nonsequential signalsacquired from the surface of the test strip are compared.

In an embodiment, the first position is a sample receiving zone. In anembodiment, the second position is a test zone. In an embodiment, thesecond position is a control zone. In an embodiment, the first positionis a sample receiving zone and the second position is a test zone. In anembodiment, the first position is a sample receiving zone and the secondposition is a control zone.

Additional features and advantages are described herein, and will beapparent from, the following Detailed Description and figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows images depicting the transit of a wavefront from a liquidsample across the surface of a lateral flow assay test strip. The liquidtraversed the test strip from left to right. An exemplary method todetermine the wavefront position may be to find the point(s) where thedifference plot (right) is larger than a given threshold (e.g., 0.05)and then find the right-most point where the signal is above this level.

DETAILED DESCRIPTION

The present disclosure provides methods and devices for determining thewavefront position of a liquid (e.g. a liquid test sample) on thesurface of a lateral flow assay. Variations in flow rate due tovariability in the assay membrane or differences in sample viscosity mayaffect the length of time that an analyte is in contact with boundantibody on the assay surface (e.g., the capture line) and thus mayaffect the amount of analyte detected by the assay. As such, it isadvantageous to determine the wavefront position of a liquid sample toobtain accurate measurements from the lateral flow assay. Such methodsof the present disclosure may comprise placing a liquid on the surfaceof the test strip; and acquiring one or more signals from the surface ofthe test strip at one or more times, comparing the one or more acquiredsignals to a threshold, wherein the wavefront position is a position onthe surface of the test strip where a signal is greater than or lessthan a threshold (e.g., fixed or dynamic threshold). The wavefrontposition may be employed to determine the transit time of the liquidsample to traverse from a first position on the surface of the lateralflow assay to a second position on the surface of the lateral flow assayincluding, for example, the length of time required for the liquidsample to traverse a first position where the sample comprising ananalyte is applied to the assay surface (e.g., a sample pad) to a secondposition where the analyte is captured by an antibody bound to thesurface of the assay surface (e.g., test line). Additionally, themethods of the present disclosure may be used to determine the transittime of a liquid sample to traverse the entire surface of the assay teststrip.

The present disclosure provides methods for measuring the flow rate of aliquid (e.g., a liquid test sample) on the surface of an assay teststrip. The sample flow rate may be determined by acquiring at least twoimages from the surface of the lateral flow assay, and calculating thedistance that the wavefront has traversed between the acquisition of atleast a first and a second image. In the case of left to right flow, thewavefront position is the right most position where the greatestdifference exists between two acquired images. The flow rate may then becalculated by dividing the distance that the wavefront has traveled bythe time elapsed between the acquisition of the first and the secondsignal.

In an exemplary method, an image (e.g., a frame) may be acquired fromthe surface of the assay with an imaging-based detector at regular timeintervals (e.g., every second). Each frame F(n) may be compared withframe F(n−1) and the difference in pixels between frames calculated.This difference is the spatial derivative, representing how much eachpixel has changed. Positions on the assay surface with the largestchange are the positions that have become wet since the acquisition ofthe previous frame, and positions with little or no change are thosepositions that have stayed dry or stayed wet. This difference can becalculated by subtracting the two consecutive frames F(n)−F(n−1), or bydividing the two frames F(n)/F(n−1). Images compared do not need to beconsecutive. Any two frames that have different acquisition times may becompared. In addition, frames could be compared by also recording theirrespective acquisition times, and then calculating the derivative asfollows: dF/dt=[F(n)−F(n−1)]/[T(n)−T(n−1)], where F(n) is the nth frame,and T(n) is the time that the nth frame was acquired. These calculationscan be made using the full 2-D image, or a 1-D representation of theimage after it has been summed or average over each column.

The methods of the present disclosure may be used to determine thetransit time for a liquid sample to traverse a distance between twopositions on the surface of the assay including, for example, the entiresurface of the assay.

The methods of the present disclosure are preferably used with animmunoassay device. One or more analytes bound to an antibody on thesurface of the immunoassay device may be detected and subsequentlyquantitated.

Exemplary assays contemplated for use with the methods of the presentdisclosure include lateral flow assay test strips. Lateral flow assaytest strips may comprise a membrane system that forms a single fluidflow pathway along the test strip. The membrane system may include oneor more components that act as a solid support for immunoreactions. Forexample, porous, bibulous or absorbent materials may be placed on astrip such that they partially overlap, or a single material can beused, in order to conduct liquid along the strip. The membrane materialsmay be supported on a backing, such as a plastic backing. In a preferredembodiment, the test strip includes a glass fiber pad, a nitrocellulosestrip and an absorbent cellulose paper strip supported on a plasticbacking.

Antibodies that react with the target analyte and/or a detectable labelsystem are immobilized on the solid support. The antibodies may be boundto the test strip by adsorption, ionic binding, van der Waalsadsorption, electrostatic binding, or by covalent binding, by using acoupling agent, such as glutaraldehyde. For example, the antibodies maybe applied to the conjugate pad and nitrocellulose strip using standarddispensing methods, such as a syringe pump, air brush, ceramic pistonpump or drop-on-demand dispenser. In a preferred embodiment, avolumetric ceramic piston pump dispenser may be used to stripeantibodies that bind the analyte of interest, including a labeledantibody conjugate, onto a glass fiber conjugate pad and anitrocellulose strip. The test strip may or may not be otherwisetreated, for example, with sugar to facilitate mobility along the teststrip or with water-soluble non-immune animal proteins, such asalbumins, including bovine serum albumin (BSA), other animal proteins,water-soluble polyamino acids, or casein to block non-specific bindingsites.

Any antibody, including polyclonal or monoclonal antibodies, or anyfragment thereof, such as the Fab fragment, that binds the analyte ofinterest, is contemplated for use herein.

An antibody conjugate containing a detectable label may be used to bindthe analyte of interest. The detectable label used in the antibodyconjugate may be any physical or chemical label capable of beingdetected on a solid support using a reader, preferably a reflectancereader, and capable of being used to distinguish the reagents to bedetected from other compounds and materials in the assay.

Suitable antibody labels are well known to those of skill in the art andinclude, but are not limited to, enzyme-substrate combinations thatproduce color upon reaction, colored particles, such as latex particles,colloidal metal or metal or carbon sol labels, fluorescent labels, andliposome or polymer sacs, which are detected due to aggregation of thelabel. In an embodiment, colloidal gold is used in the labeled antibodyconjugate. The label may be derivatized for linking antibodies, such asby attaching functional groups, such as carboxyl groups to the surfaceof a particle to permit covalent attachment of antibodies. Antibodiesmay be conjugated to the label using well known coupling methods.

The assay test strip may be any conventional lateral flow assay teststrip such as disclosed in EP 291194 or U.S. Pat. No. 6,352,862. Thetest strip may comprise a porous carrier containing a particulatelabeled specific binding reagent and an unlabelled specific bindingreagent. The light sources and corresponding photodetectors arepreferably so aligned such that during use, light from the light sourceor sources falls upon the respective zones on the porous carrier and isreflected or transmitted to the respective photodetectors. Thephotodetectors generate a current roughly proportional to the amount oflight falling upon it which is then fed through a resistor to generate avoltage. The amount of light reaching the photodetector depends upon theamount of colored particulate label present and therefore the amount ofanalyte. Thus the amount of analyte present in the sample may bedetermined. This method of optically determining the analyteconcentration is described more fully in EP 653625.

A sample may include, for example, anything which may contain an analyteof interest. The sample may be a biological sample, such as a biologicalfluid or a biological tissue. Examples of biological fluids includeurine, blood, plasma, serum, saliva, semen, stool, sputum, cerebralspinal fluid, tears, mucus, amniotic fluid or the like. Biologicaltissues are aggregate of cells, usually of a particular kind togetherwith their intercellular substance that form one of the structuralmaterials of a human, animal, plant, bacterial, fungal or viralstructure, including connective, epithelium, muscle and nerve tissues.Examples of biological tissues also include organs, tumors, lymph nodes,arteries and individual cells.

A fluid sample (e.g., biological fluid) may refer to a materialsuspected of containing the analyte(s) of interest, which material hassufficient fluidity to flow through an immunoassay device in accordanceherewith. The fluid sample can be used as obtained directly from thesource or following a pretreatment so as to modify its character. Suchsamples can include human, animal or man-made samples. The sample can beprepared in any convenient medium which does not interfere with theassay.

The fluid sample can be derived from any source, such as a physiologicalfluid, including blood, serum, plasma, saliva, sputum, ocular lensfluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid,peritoneal fluid, transdermal exudates, pharyngeal exudates,bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid,semen, cervical mucus, vaginal or urethral secretions, amniotic fluid,and the like. Herein, fluid homogenates of cellular tissues such as, forexample, hair, skin and nail scrapings, meat extracts and skins offruits and nuts are also considered biological fluids. Pretreatment mayinvolve preparing plasma from blood, diluting viscous fluids, and thelike. Methods of treatment can involve filtration, distillation,separation, concentration, inactivation of interfering components, andthe addition of reagents. Besides physiological fluids, other samplescan be used such as water, food products, soil extracts, and the likefor the performance of industrial, environmental, or food productionassays as well as diagnostic assays. In addition, a solid materialsuspected of containing the analyte can be used as the test sample onceit is modified to form a liquid medium or to release the analyte.

Exemplary lateral flow devices include those described in U.S. Pat. Nos.4,818,677, 4,943,522, 5,096,837 (RE 35,306), 5,096,837, 5,118,428,5,118,630, 5,221,616, 5,223,220, 5,225,328, 5,415,994, 5,434,057,5,521,102, 5,536,646, 5,541,069, 5,686,315, 5,763,262, 5,766,961,5,770,460, 5,773,234, 5,786,220, 5,804,452, 5,814,455, 5939,331,6,306,642.

A sample may include, for example, anything which may contain ananalyte. The sample may be a biological sample, such as a biologicalfluid or a biological tissue. Examples of biological fluids includeurine, blood, plasma, serum, saliva, semen, stool, sputum, cerebralspinal fluid, tears, mucus, amniotic fluid or the like. Biologicaltissues are aggregate of cells, usually of a particular kind togetherwith their intercellular substance that form one of the structuralmaterials of a human, animal, plant, bacterial, fungal or viralstructure, including connective, epithelium, muscle and nerve tissues.Examples of biological tissues also include organs, tumors, lymph nodes,arteries and individual cell(s). A liquid sample may refer to a materialsuspected of containing the analyte(s) of interest, which material hassufficient fluidity to flow through an immunoassay device in accordanceherewith. The fluid sample can be used as obtained directly from thesource or following a pretreatment so as to modify its character. Suchsamples can include human, animal or man-made samples. The sample can beprepared in any convenient medium which does not interfere with theassay. Typically, the sample is an aqueous solution or biological fluidas described in more detail below.

The fluid sample can be derived from any source, such as a physiologicalfluid, including blood, serum, plasma, saliva, sputum, ocular lensfluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid,peritoneal fluid, transdermal exudates, pharyngeal exudates,bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid,semen, cervical mucus, vaginal or urethral secretions, amniotic fluid,and the like. Herein, fluid homogenates of cellular tissues such as, forexample, hair, skin and nail scrapings, meat extracts and skins offruits and nuts are also considered biological fluids. Pretreatment mayinvolve preparing plasma from blood, diluting viscous fluids, and thelike. Methods of treatment can involve filtration, distillation,separation, concentration, inactivation of interfering components, andthe addition of reagents. Besides physiological fluids, other samplescan be used such as water, food products, soil extracts, and the likefor the performance of industrial, environmental, or food productionassays as well as diagnostic assays. In addition, a solid materialsuspected of containing the analyte can be used as the test sample onceit is modified to form a liquid medium or to release the analyte.

An analyte can be any substance for which there exists a naturallyoccurring analyte specific binding member or for which ananalyte-specific binding member can be prepared. e.g., carbohydrate andlectin, hormone and receptor, complementary nucleic acids, and the like.Further, possible analytes include virtually any compound, composition,aggregation, or other substance which may be immunologically detected.That is, the analyte, or portion thereof, will be antigenic or haptenichaving at least one determinant site, or will be a member of a naturallyoccurring binding pair.

Analytes include, but are not limited to, toxins, organic compounds,proteins, peptides, microorganisms, bacteria, viruses, amino acids,nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs(including those administered for therapeutic purposes as well as thoseadministered for illicit purposes), pollutants, pesticides, andmetabolites of or antibodies to any of the above substances. The termanalyte also includes any antigenic substances, haptens, antibodies,macromolecules, and combinations thereof (see, e.g., U.S. Pat. Nos.4,366,241; 4,299,916; 4,275,149; and 4,806,311).

In an embodiment, a sample receiving zone on the surface of a lateralflow assay test strip accepts a fluid sample that may contain one ormore analytes of interest. In an embodiment, the sample receiving zoneis dipped into a fluid sample. A label zone is located downstream of thesample receiving zone, and contains one or more mobile label reagentsthat recognize, or are capable of binding the analytes of interest.Further, a test region may be disposed downstream from the label zone,and contains test and control zones. The test zone(s) generally containmeans which permit the restraint of a particular analyte of interest ineach test zone. Frequently, the means included in the test zone(s)comprise an immobilized capture reagent that binds to the analyte ofinterest. Generally the immobilized capture reagent specifically bindsto the analyte of interest. Thus, as the fluid sample flows along thematrix, the analyte of interest will first bind with a mobilizable labelreagent in the label zone, and then become restrained in the test zone.

In an embodiment, the sample receiving zone may be comprised of anabsorbent application pad. Suitable materials for manufacturingabsorbent application pads include, but are not limited to, hydrophilicpolyethylene materials or pads, acrylic fiber, glass fiber, filter paperor pads, desiccated paper, paper pulp, fabric, and the like. Forexample, the sample receiving zone may be comprised of a material suchas a nonwoven spunlaced acrylic fiber.

The sample receiving zone may be comprised of any material from whichthe fluid sample can pass to the label zone. Further, the absorbentapplication pad can be constructed to act as a filter for cellularcomponents, hormones, particulate, and other certain substances that mayoccur in the fluid sample. Application pad materials suitable for use bythe present invention also include those application pad materialsdisclosed in U.S. Pat. No. 5,075,078.

In a further embodiment, the sample receiving zone may be comprised ofan additional sample application member (e.g., a wick). Thus, in oneaspect, the sample receiving zone can comprise a sample application padas well as a sample application member. Often the sample applicationmember is comprised of a material that readily absorbs any of a varietyof fluid samples contemplated herein, and remains robust in physicalform. Frequently, the sample application member is comprised of amaterial such as white bonded polyester fiber. Moreover, the sampleapplication member, if present, is positioned in fluid-flow contact witha sample application pad.

In an embodiment, the label zone material may be treated with labeledsolution that includes material-blocking and label-stabilizing agents.Blocking agents include, for example, bovine serum albumin (BSA),methylated BSA, casein and nonfat dry milk. Stabilizing agents arereadily available and well known in the art, and may be used, forexample, to stabilize labeled reagents.

The label zone may contain a labeled reagent, often comprising one ormore labeled reagents. In many of the presently contemplatedembodiments, multiple types of labeled reagents are incorporated in thelabel zone such that they may permeate together with a fluid samplecontacted with the device. These multiple types of labeled reagent canbe analyte specific or control reagents and may have differentdetectable characteristics (e.g., different colors) such that onelabeled reagent can be differentiated from another labeled reagent ifutilized in the same device. As the labeled reagents are frequentlybound to a specific analyte of interest subsequent to fluid sample flowthrough the label zone, differential detection of labeled reagentshaving different specificities (including analyte specific and controllabeled reagents) may be a desirable attribute. However, frequently, theability to differentially detect the labeled reagents having differentspecificities based on the label component alone is not necessary due tothe presence of test and control zones in the device, which allow forthe accumulation of labeled reagent in designated zones.

The labeling zone may also include control-type reagents. These labeledcontrol reagents often comprise detectable moieties that will not becomerestrained in the test zones and that are carried through to the testregion and control zone(s) by fluid sample flow through the device. In afrequent embodiment, these detectable moieties are coupled to a memberof a specific binding pair to form a control conjugate which can then berestrained in a separate control zone of the test region by acorresponding member of the specific binding pair to verify that theflow of liquid is as expected. The visible moieties used in the labeledcontrol reagents may be the same or different color, or of the same ordifferent type, as those used in the analyte of interest specificlabeled reagents. If different colors are used, ease of observing theresults may be enhanced.

The test region may include a control zone for verification that thesample flow is as expected. Each of the control zones comprise aspatially distinct region that often includes an immobilized member of aspecific binding pair which reacts with a labeled control reagent. In anoccasional embodiment, the procedural control zone contains an authenticsample of the analyte of interest, or a fragment thereof. In thisembodiment, one type of labeled reagent can be utilized, wherein fluidsample transports the labeled reagent to the test and control zones; andthe labeled reagent not bound to an analyte of interest will then bindto the authentic sample of the analyte of interest positioned in thecontrol zone. In another embodiment, the control line contains antibodythat is specific for, or otherwise provides for the immobilization of,the labeled reagent. In operation, a labeled reagent is restrained ineach of the one or more control zones, even when any or all the analytesof interest are absent from the test sample.

Since the devices of the present invention may incorporate one or morecontrol zones, the labeled control reagent and their correspondingcontrol zones are preferably developed such that each control zone willbecome visible with a desired intensity for all control zones afterfluid sample is contacted with the device, regardless of the presence orabsence of one or more analytes of interest. In one embodiment, a singlelabeled control reagent will be captured by each of the control zones onthe test strip. Frequently, such a labeled control reagent will bedeposited onto or in the label zone in an amount exceeding the capacityof the total binding capacity of the combined control zones if multiplecontrol zones are present. Accordingly, the amount of capture reagentspecific for the control label can be deposited in an amount that allowsfor the generation of desired signal intensity in the one or morecontrol zones, and allows each of the control zones to restrain adesired amount of labeled control-reagent. At the completion of anassay, each of the control zones preferably provide a desired and/orpre-designed signal (in intensity and form).

In an embodiment, each control zone will be specific for a uniquecontrol reagent. In this embodiment, the label zone may include multipleand different labeled control reagents, equaling the number of controlzones in the assay, or a related variation. Wherein each of the labeledcontrol reagents may become restrained in one or more pre-determined andspecific control zone(s). These labeled control reagents can provide thesame detectable signal (e.g., be of the same color) or providedistinguishable detectable signals (e.g., have different colored labelsor other detection systems) upon accumulation in the control zone(s).

In an embodiment, the labeled control reagent comprises a detectablemoiety coupled to a member of a specific binding pair. Typically, alabeled control reagent is chosen to be different from the reagent thatis recognized by the means which are capable of restraining an analyteof interest in the test zone. Further, the labeled control reagent isgenerally not specific for the analyte. In a frequent embodiment, thelabeled control reagent is capable of binding the corresponding memberof a specific binding pair or control capture partner that isimmobilized on or in the control zone. Thus the labeled control reagentis directly restrained in the control zone.

The use of a control zone is helpful in that appearance of a signal inthe control zone indicates the time at which the test result can beread, even for a negative result. Thus, when the expected signal appearsin the control line, the presence or absence of a signal in a test zonecan be noted.

Test zones of the present description include means that permit therestraint of an analyte of interest. Frequently, test zones of thepresent description include a ligand that is capable of specificallybinding to an analyte of interest. Alternatively, test zones of thepresent description include a ligand that is capable of specificallybinding the labeled reagent bound to an analyte of interest. Inpractice, a labeled test reagent binds an analyte of interest present ina fluid sample after contact of the sample with a representative deviceand flow of the fluid sample into and through the label zone.Thereafter, the fluid sample containing the labeled analyte progressesto a test zone and becomes restrained in the test zone. The accumulationof labeled analyte in the test zone produces a detectable signal.Devices may incorporate one or more test zones, each of which is capableof restraining different analytes, if present, in a fluid sample. Thus,in representative embodiments two, three, four, five or more (labeled)analytes of interest can be restrained in a single or different testzones, and thereby detected, in a single device.

The present devices may optionally further comprise an absorbent zonethat acts to absorb excess sample after the sample migrates through thetest region. The absorbent zone, when present lies in fluid flow contactwith the test region. This fluid flow contact can comprise anoverlapping, abutting or interlaced type of contact. In an occasionalembodiment, a control region (end of assay indicator) is provided in theabsorbent zone to indicate when the assay is complete. In thisembodiment, specialized reagents are utilized, such as pH sensitivereagents (such as bromocresol green), to indicate when the fluid samplehas permeated past all of the test and control zones.

The test strip optionally may be contained within a housing forinsertion into the reflectance reader. The housing may be made ofplastic or other inert material that does not interfere with the assayprocedure.

The lateral flow assay test strip may be suited for use with a readingdevice that comprises one or more of the following: a central processingunit (CPU) or microcontroller; two or more LED's; two or morephotodiodes; a power source; and associated electrical circuitry. Thepower source may comprise a battery or any other suitable power source(e.g. a photovoltaic cell). The CPU will typically be programmed so asto determine whether the calculated rate and/or extent of progress ofthe liquid sample is within predetermined limits.

Conveniently the assay result reading device will comprise some mannerof indicating the result of the assay to a user. This may take the form,for example, of an audible or visible signal. Desirably the device willcomprise a visual display to display the assay result. This may simplytake the form of one or more LED's or other light sources, such thatillumination of a particular light source or combination of lightsources conveys the necessary information to the user. Alternatively thedevice may be provided with an alphanumeric or other display, such as anLCD. In addition, or as an alternative, to displaying the assay result,the device may also display or indicate in some other way to the userwhether the calculated rate and/or extent of progress of the liquidsample is within the predetermined acceptable limits, and thus whetheror not the result of the particular assay should be disregarded. If thereading device determines that a particular assay result should bedisregarded it may prompt the user to repeat the assay.

Any device which is compatible for use with an assay test strip,preferably a reflectance reader, for determining the assay result iscontemplated for use herein. Such test strip devices as are known tothose of skill in the art (see, e.g., U.S. Pat. Nos. 5,658,801,5,656,502, 5,591,645, 5,500,375, 5,252,459, 5,132,097). Reflectance andother readers, including densitometers and transmittance readers, areknown to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,598,007,5,132,097, 5,094,955, 4,267,261, 5,118,183, 5,661,563, 4,647,544,4,197,088, 4,666,309, 5,457,313, 3,905,767, 5,198,369, 4,400,353).

While the present disclosure has been described and illustrated hereinby references to various specific materials, procedures and examples, itis understood that the disclosure is not restricted to the particularcombinations of material and procedures selected for that purpose.Numerous variations of such details can be implied as will beappreciated by those skilled in the art. It is intended that thespecification and examples be considered as exemplary, only, with thetrue scope and spirit of the disclosure being indicated by the followingclaims. All references, patents, and patent applications referred to inthis application are herein incorporated by reference in their entirety.

The invention claimed is:
 1. A system for determining a rate of progressof a liquid to flow from a first position to a second position along alength of a test strip, said system comprising: the test strip; and areader device comprising: one or more light source or sources positionedto emit light onto a surface of the test strip; photodetectorspositioned to detect the light after reflection from the surface of thetest strip, the photodetectors configured to: acquire a first image ofthe surface of the test strip at a first time, the first timecorresponding to a starting time at which the sample is placed in aliquid receiving zone of the test strip, the liquid receiving zonecomprising the first position, the first image comprising a firstplurality of pixels; acquire a second image of the surface of the teststrip at a second time after the first time, the second image comprisinga second plurality of pixels; and a central processing unit ormicrocontroller configured to: determine a result indicated by the teststrip; generate a difference plot representing level of change valuesalong the length of the test strip, a particular level of change valuecomputed between one of the first plurality of pixels and acorresponding one of the second plurality of pixels; identify a point onthe difference plot farthest along the length of the test strip from theliquid receiving zone where the level of change value is above athreshold; determine a wavefront position based on a location along thelength of the test strip corresponding to the point; identify a distancebetween the first position and the wavefront position; calculate therate of progress of the sample across the test strip based on theidentified distance and a time elapsed between the second time and thefirst time; determine whether the result is accurate based on whetherthe rate of progress is within a predetermined limit; in response todetermining that the rate of progress is not within the predeterminedlimit, disregard the result of the test strip and output a notificationassociated with disregarding the result of the test strip; and inresponse to determining that the rate of progress is within thepredetermined limit, output the result of the test strip.
 2. The systemof claim 1, wherein the central processing unit is configured tosubtract values of the first and second plurality of pixels from aconstant prior to comparing said values to the threshold value.
 3. Thesystem of claim 1, wherein the central processing unit is configured todivide values of the first and second plurality of pixels by a constantprior to comparing said values to the threshold value.
 4. The system ofclaim 1, wherein the central processing unit is configured to comparevalues of corresponding pixels of the first and second plurality ofpixels with each other prior to comparing said values to the thresholdvalue.
 5. The system of claim 1, wherein the central processing unit isconfigured to subtract values of corresponding pixels of the first andsecond plurality of pixels prior to comparing said values to thethreshold value.
 6. The system of claim 1, wherein the centralprocessing unit is configured to divide a value of each of the firstplurality of pixels by a value of a corresponding one of the secondplurality of pixels prior to comparing said values to the thresholdvalue.
 7. The system of claim 1, wherein the central processing unit isconfigured to acquire the first image during insertion of the test stripinto the reader device.
 8. The system of claim 1, wherein the test stripis a lateral flow assay test strip.
 9. The system of claim 1, whereinthe liquid is a test sample.
 10. The system of claim 1, wherein thecentral processing unit is configured to acquire a plurality of imagesof the surface of the test strip at regular intervals, wherein the firstimage and the second image are sequential images of the plurality ofimages.
 11. The system of claim 1, wherein the central processing unitis configured to acquire a plurality of images of the surface of thetest strip at regular intervals, wherein the first image and the secondimage are non-sequential images of the plurality of images.
 12. Thesystem of claim 1, wherein the test strip comprises a test zone havingan immobilized capture reagent that binds to an analyte of interest inthe liquid.
 13. The system of claim 12, wherein the predetermined limitfor the rate of progress is determined at least partly based on a lengthof time required for the analyte to be in contact with the immobilizedcapture reagent to detect a threshold amount of the analyte.
 14. Thesystem of claim 12, wherein the central processing unit is configuredto: acquire an additional image of the test zone after binding of theanalyte of interest with the immobilized capture reagent; and determinethe result of the test strip based on an amount of light detected fromthe test zone.
 15. The system of claim 14, wherein the centralprocessing unit is configured to determine the result by determining aconcentration of the analyte of interest in the liquid.
 16. The systemof claim 12, wherein the second position comprises the test zone. 17.The system of claim 1, wherein the second position comprises an end ofthe test strip opposite the liquid receiving zone.